National University Medical Institutes

Activation of the peroxisome proliferator-activated receptor-g (PPARg has been identified as an approach for inducing differentiation and inhibiting proliferation of a variety of cancer cells. This is particularly true for breast cancers where the expression of PPARg is many folds higher than normal breast epithelium, independent of ER, p53, or HER2/neu status. In addition, cells’ exposure to synthetic PPARg ligands, such as proglitazone or rosiglitazone exert anti-tumor activity via cell growth inhibition and cellular differentiation, however, the precise mechanism and genes involved in this anticancer activity of PPARg ligands are poorly understood.

Assess the role of PPARg dependent production of reactive oxygen species (ROS) in the repression of NHE1 gene expression
Recent studies showed that rosiglitazone at concentrations of 5-20mM decreased survival of glioma cells without affecting primary astrocytes by a mechanism mediated by an increase in mitochondrial-generated ROS. In addition, in a mouse monocyte/macrophage cell line, ligand-activated PPARg downregulates expression of p47 phagocyte oxidase (p47phox), one important component of the NAD(P)H oxidase enzyme complex, thereby lowering intracellular O2.- This is interesting to us because we have shown that NHE1 expression is down-regulated with increasing concentrations of H2O2, a priori inhibiting cell growth. Conversely, a decrease in intracellular O2.- down-regulated NHE1 expression whereby cells are sensitized to apoptotic triggers. Hence, we propose to first assess if incubation of MCF-7, MDA-MB-231, and T47D cells with ligands of PPARg could influence intracellular ROS levels.

Assess if growth arrest of tumor cells upon exposure to PPARg ligands is dependent on the repression of NHE1 gene expression

A recent study showed that the PPARg ligand, troglitazone induces cellular acidosis by inhibiting NHE1 activity in breast carcinoma-derived cell line. We showed in a separate study that a decrease in NHE1 pump affinity (set point) and intracellular acidification corroborated to the observed decrease in NHE1 protein levels following H2O2 treatment. More importantly, in the same manuscript, we show a decrease in NHE1 protein level leads to a more sustainable decrease in intracellular pH. Because Putney and Barber has shown intracellular pH (via NHE1 activity) times cells’ G2/M entry and transition, in this study, we would determine if intracellular pH and NHE pump affinity decreases following 15d-PGJ2 treatment in MCF-7, MDA-MB-231, and T47D cells.